PROJECT SUMMARY Human embryonic stem cells (hESCs) are an invaluable tool for human disease modeling and the field of regenerative medicine. Human ESCs behave like stem cells in culture, having the capacity to divide in culture (self-renew) and the ability to produce different cell types (differentiation). However, there is still much to learn about the mechanisms and genetic regulators that control hESC growth and differentiation. An understanding of these biological processes will significantly impact their application in drug discovery for the treatment of human diseases. Many biological processes in mammalian cells are regulated through modification of target proteins by a small, highly conserved protein called ubiquitin. The covalent attachment of ubiquitin to proteins leads to their degradation, a mechanism used for regulating cell function. This process, known as ubiquitination, is also essential for controlling cell differentiation. Our laboratory recently identified a novel key regulator that is required for the generation of neural crest cells when hESC are induced to differentiate in vitro. This process requires the ubiquitination of critical cellular components. Since neural crest cells are a transient and migratory cell population essential for craniofacial development, this finding helped identify a key step in the determination of neural crest cell fate. The goal of this work is to further understand the role and the mechanisms by which the ubiquitin system influences neural crest cell specification in hESCs. To do this, I will use an unbiased approach to abrogate or activate ubiquitination enzymes in cells and determine how such perturbations affect neural crest cell differentiation. We hope that dissection of these complex biochemical pathways will provide a means to manipulate neural crest cells in the laboratory, a critical step in using this culture system as a disease model. More importantly, this study will help identify ubiquitination components that are essential for maintaining the neural crest state. By manipulating the activity of key proteins via ubiquitination, it may be possible to understand the mechanisms and/or pathways that dictate neural crest cell growth and differentiation. With this information, synthetic compounds that target ubiquitin enzymes and/or their substrates may selectively prevent neural crest cell growth and serve as therapeutic agents to treat neural crest-derived tumors.